C07K 14/195 - Peptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from bacteria
C12Q 1/00 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urineTesting involving biospecific ligand binding methodsImmunological testing
11.
METHOD OF CHARACTERISING POLYPEPTIDES USING A NANOPORE
Provided herein are methods of characterising a target polypeptide as it moves with respect to a nanopore. Also provided are related kits, systems and apparatuses for carrying out such methods.
The invention relates generally to a method of detecting and/or analysing target polymers, especially target polynucleotides, using a biological pore. The invention also relates to a novel system for carrying out the method. The method has many uses. In particular, the method may be used for diagnosis, detection of polymorphisms and V(D)J repertoire analysis.
The invention relates to mutant forms of Cytotoxin K. The invention also relates to methods of analyte detection and characterisation using Cytotoxin K, together with devices and kits for carrying out such methods.
IMPERIAL COLLEGE INNOVATIONS LIMITED (United Kingdom)
Inventor
Heron, Andrew John
Gutierrez, Richard Alexander
Edel, Joshua
Ivanov, Aleksandar
Koch, Caroline
Xue, Liang
Reilly-O'Donnell, Benedict
Abstract
A method for detecting multiple molecules in a sample, the method comprising: (a) contacting the sample with a carrier and a nanopore, wherein the carrier comprises a single-stranded leader, an identifier region and a molecule-binding region specific for a molecule to be detected, and wherein a motor protein is bound to the carrier such that it can control the movement of the identifier region within the nanopore; (b) taking one or more optical or electrical measurements as a carrier moves within the nanopore to characterise the identifier region and to determine whether or not the molecule is bound to the molecule-binding region.
Provided herein is a method of chemically modifying a polypeptide or polynucleotide monomer. The monomer may be a monomer of a protein pore such as a monomeric or oligomeric protein pore. Also disclosed are related monomers and oligomers and methods of using the disclosed pores in characterising analytes.
Provided herein is a method of characterising a target polynucleotide as it moves with respect to a nanopore using a motor protein. Also provided are polynucleotide adapters and kits comprising such adapters. The methods, kits and adapters find use in characterising polynucleotides, for example in sequencing.
Provided herein are methods of characterising a target polypeptide as it moves with respect to a nanopore. Also provided are related kits, systems and apparatuses for carrying out such methods.
The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.
Provided herein is a method of loading a motor protein onto a polynucleotide adapter. Also provided are polynucleotide adapters and kits comprising such adapters. The adapters find use in characterising analytes such as polynucleotides in methods in which the polynucleotide moves in respect of a nanopore.
A modified portal protein of a bacteriophage DNA packaging motor, wherein the modified portal protein is capable of direct insertion into a membrane and wherein the portal protein is modified compared to the wild type portal protein such that one or more amino acid residues on the outer surface of the portal protein is substituted by one or more other amino acid residue, and/or wherein a one or more amino acid residue is inserted on the outer surface of the portal protein so as to alter the outer surface hydrophobicity of the modified portal protein compared to the wild type portal protein.
A signal derived from a polymer comprising a series of polymer units during translocation of the polymer with respect to a nanopore is analysed using a machine learning technique. A series of weight distributions is output, each comprising weights in respect of transitions between labels over a set of labels representing the possible types of polymer unit in the polymer. An estimate of the series of polymer units from the weight distributions. Use of weights in respect of the transitions increases the accuracy of the technique.
A system for characterising a target polynucleotide, the system comprising a membrane and a pore complex; wherein the pore complex comprises: (i) a nanopore located in the membrane, and (ii) an auxiliary protein or peptide attached to the nanopore; wherein the nanopore and the auxiliary protein or peptide together form a continuous channel across the membrane, the channel comprising a first constriction region and a second constriction region; wherein the first constriction region is formed by a portion of the nanopore, and wherein the second constriction region is formed by at least a portion of the auxiliary protein or peptide.
The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.
The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.
The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. ine invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.
A method for selectively modifying a target polynucleotide in a sample of polynucleotides, the method comprising contacting a sample of polynucleotides with a guide polynucleotide that binds to a sequence in the target polynucleotide and a polynucleotide-guided effector protein such that the polynucleotide-guided effector protein cuts the target polynucleotide to produce a cut end comprising an overhang; and attaching an adapter to the cut end in the target polynucleotide.
A component (8) adapted to engage with a receiver (6) has an array of contact pads (100) to removably connect with a corresponding array of connectors (18) on the receiver (6). Each contact pad (100) of the array is electrically connected to the electrode (26) of a corresponding recess or well (28) that is part of a sensor, wherein a membrane is formable across each recess. A conductive grid (102) is configured between the contact pads (100) of the array, to inhibit an electrostatic discharge (BSD) conducting across the recesses or wells and/or direct an BSD away from the recesses or wells.
The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.
A method for selecting polynucleotides, the method comprising: allowing a nucleic acid handling enzyme to move along multiple polynucleotides in a sample for a defined time period, wherein the enzyme is loaded onto each of the multiple polynucleotides and wherein one or more molecule of the enzyme moves along each of the multiple polynucleotides; and selecting polynucleotides based on whether or not the enzyme reaches the end of and/or unbinds from the polynucleotides in the defined time period.
Droplet interfaces are formed between droplets in an electro-wetting device comprising an array of actuation electrodes. Actuation signals are applied to selected actuation electrodes to place the droplets into an energised state in which the shape of the droplets is modified compared to a shape of the droplets in a lower energy state and to bring the two droplets into proximity. The actuation signals are then changed to lower the energy of the droplets into the lower energy state so that the droplets relax into the gap and the two droplets contact each other thereby forming a droplet interface. The use of sensing electrodes in the device permit electrical current measurements across the droplet interface. The sensing electrodes can be used for either (i) applying a reference signal during droplet actuation or (ii) recording electrical current measurements.
A microfluidic device for analysing a test liquid comprises: a sensor (235), such as a membrane provided with nanopores, provided in a sensing chamber (237); a sensing chamber inlet channel (261) and a sensing chamber outlet channel (262), each connecting to the sensing chamber for respectively passing liquid into and out of the sensing chamber, and a reservoir (233) forming a sample input port to the microfluidic device, the reservoir being in fluid communication with the sensing chamber inlet channel (261); a liquid collection channel (232); a barrier (231) between an end of the sensing chamber outlet channel (262) and the liquid collection channel (232); a first seal (251), covering the sample input port; a second seal (252), covering the end of the sensing chamber outlet channel (262), thereby preventing liquid from flowing from the sensing chamber (237), over the barrier (231), into the liquid collection channel (232); wherein the microfluidic device is filled with a liquid from the first seal (251) at the sample input port to the second seal (252), such that the sensor (235) is covered by liquid and unexposed to a gas or gas/liquid interface; and wherein the first and second seals (251, 252) are removable to cause the liquid to flow between the reservoir and the end of the sensing chamber outlet and over the barrier.
A kit comprising first and second components the first component comprising a first array of electrical connectors; two substantially parallel lateral walls on either side of the first array; two rails between the first array and the lateral walls, one rail on either side of the first array, each rail extending along a length of the first array, a front tip; a front contact point; and an overhang to receive the second component; the second component comprising a second array of electrical connectors, to connect to the first array; a front end fitting a width between the parallel lateral walls, lateral sides, each comprising a rail relief allowing the sides to fit around the two rails, forming a shoulder by the front end of each rail relief; wherein the first and second component are configured to slidingly connect the second array to the first array in a correct position.
The present invention relates to novel protein pores and their uses in analyte detection and characterisation. The invention particularly relates to an isolated pore complex formed by a CsgG-like pore and a modified CsgF peptide, or a homologue or mutant thereof, thereby incorporating an additional channel constriction or reader head in the nanopore. The invention further relates to a transmembrane pore complex and methods for production of the pore complex and for use in molecular sensing and nucleic acid sequencing applications.
Provided is a method of characterising a polynucleotide using a transmembrane pore, wherein the pore is a double pore comprising a first Csg G pore, or a homologue thereof, and a second CsgG pore, or a homologue thereof.
Provided herein relate to modified or mutant forms of secretin and compositions comprising the same. In particular, the modified or mutant forms of secretin permits efficient capture and/or translocation of an analyte through the modified or mutant secretin nanopores. Methods for using unmodified secretin or the modified or mutant forms of secretin and compositions, for example, for characterizing an analyte, e.g., a target polynucleotide, are also provided.
The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.
B01J 19/00 - Chemical, physical or physico-chemical processes in generalTheir relevant apparatus
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C12N 15/00 - Mutation or genetic engineeringDNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purificationUse of hosts therefor
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
Methods of characterizing an analyte using a nanopore. One aspect features methods for characterizing a double-stranded polynucleotide using a nanopore, e.g., without using a hairpin connecting a template and a complement of the double-stranded polynucleotide. Another aspect features methods for characterizing an analyte using a tag-modified nanopore with increased sensitivity and/or higher throughput. Compositions and systems including, e.g., adaptors for attachment to double-stranded polynucleotides and tag-modified nanopores, which can be used in the methods are also provided.
Apparatus and methods for controlling the insertion of a membrane channel into a membrane are disclosed. In one arrangement a first bath holds a first liquid in contact with a first surface of a membrane. A second bath holds a second liquid in contact with a second surface of the membrane. The membrane separates the first and second liquids. A first electrode contacts the first liquid. A second electrode contacts the second liquid. A driving unit applies a potential difference across the membrane via the first and second electrodes to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid. A membrane voltage reduction unit is connected in series with the membrane. The driving unit applies a driving voltage across the membrane voltage reduction unit and the membrane, the driving voltage providing the potential difference across the membrane. The membrane voltage reduction unit is configured such that a reduction in resistance through the membrane caused by insertion of a membrane channel intrinsically increases a potential difference across the membrane voltage reduction unit thereby lowering the potential difference across the membrane. The lowering of the potential difference across the membrane is sufficient to prevent or reduce promotion of insertion of a further membrane channel.
C07K 14/435 - Peptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from animalsPeptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from humans
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C07K 14/435 - Peptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from animalsPeptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from humans
The invention relates to pores having an enhanced ability to estimate the characteristics of analytes, such as the sequence of polynucleotides. The mutant pores surprisingly provide more consistent movement of a target polynucleotide with respect to, such as through, the pores. Mutant forms of CsgG sequence are shown in SEQ ID NO: 2, wherein the variant comprises K94N/Q/R/F/Y/W/L/S and retains the ability to form a pore. The invention also relates to analyte detection and characterisation using said CsgG.
C12N 15/31 - Genes encoding microbial proteins, e.g. enterotoxins
C12Q 1/00 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
G01N 27/414 - Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
G01N 33/48 - Biological material, e.g. blood, urineHaemocytometers
G01N 33/483 - Physical analysis of biological material
43.
MUTANT CSGG PORES AND THEIR USE IN POLYNUCLEOTIDE DETECTION AND CHARACTERISATION
C12M 1/34 - Measuring or testing with condition measuring or sensing means, e.g. colony counters
C12N 15/31 - Genes encoding microbial proteins, e.g. enterotoxins
C12Q 1/00 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
G01N 27/414 - Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
G01N 33/48 - Biological material, e.g. blood, urineHaemocytometers
G01N 33/483 - Physical analysis of biological material
44.
METHOD FOR CONTROLLING THE MOVEMENT OF A POLYNUCLEOTIDE THROUGH A TRANSMEMBRANE PORE
The invention relates to new methods of controlling the movement of polynucleotides through transmembrane pores. The invention also relates to new methods of characterising target polynucleotides using helicases.
The invention relates to new methods of attaching one or more polynucleotide binding proteins to a target polynucleotide. The invention also relates to new methods of characterising target polynucleotides.
C07H 21/00 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C12Q 1/6876 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
C12Q 1/689 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
46.
METHOD OF CHARACTERIZING A TARGET RIBONUCLEIC ACID (RNA) COMPRISING FORMING A COMPLEMENTARY POLYNUCLEOTIDE WHICH MOVES THROUGH A TRANSMEMBRANE PORE
The invention relates to a new method of characterising a target ribonucleic acid (RNA) involving forming a complementary polynucleotide. The method uses a transmembrane pore.
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a Dda helicase. The helicase controls the movement of the target polynucleotide through the pore. The invention also relates to modified Dda helicases which can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.
The invention relates to modified helicases with reduced unbinding from polynucleotides. The helicases can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.
A time-ordered series of measurements of a polymer made during translocation of the polymer through a nanopore are analysed. The measurements are dependent on the identity of k-mers in the nanopore, a k-mer being k polymer units of the polymer, where k is a positive integer. The method involves deriving, from the series of measurements, a feature vector of time-ordered features representing characteristics of the measurements; and determining similarity between the derived feature vector and at least one other feature vector.
A sequence of polymer units in a polymer, eg. DNA, is estimated from at least one series of measurements related to the polymer, eg. ion current as a function of translocation through a nanopore, wherein the value of each measurement is dependent on a k- mer being a group of k polymer units. A probabilistic model, especially a hidden Markov model (HMM), is provided, comprising, for a set of possible k-mers: transition weightings representing the chances of transitions from origin k-mers to destination k- mers; and emission weightings in respect of each k-mer that represent the chances of observing given values of measurements for that k-mer. The series of measurements is analysed using an analytical technique, eg. Viterbi decoding, that refers to the model and estimates at least one estimated sequence of polymer units in the polymer based on the likelihood predicted by the model of the series of measurements being produced by sequences of polymer units. In a further embodiment, different voltages are applied across the nanopore during translocation in order to improve the resolution of polymer units.
C12M 1/42 - Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic wave
C12Q 1/00 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions
C12Q 1/34 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving hydrolase
patents
